Characterization And Bioactivity Determination Of Sorghum Bicolor L.Leaf Sheath Polysaccharides:Separation And Purification Techniques

Sorghum bicolor L.（Sorghum）is a grass species that originates from Northeastern Africa.It is mainly cultivated to obtain its grain for food,animal feed,and ethanol production.Nutritional analysis of the alcohol extract of its leaf sheath has revealed the presence of carbohydrates（75.39 g/100 g）,proteins（4.87 g/100 g）and dietary fiber（50.30 g/100 g）.Studies have shown that the consumption of diets prepared with the sorghum leaf sheath provides natural antioxidants and essential fatty acids that can fight cardiovascular-related diseases.Curative interventions such as the use of pharmaceutical drugs and alternative plant medicines are increasingly being explored.Plant polysaccharides have gained attention for their promising bioactivities such as antioxidant,antimicrobial and anticancer activities.Bioactive plant polysaccharides are also being preferred for their relatively few side effects compared to conventional pharmaceuticals.Bioactive plant polysaccharides may become new leads for nutraceutical therapeutics.The general objective of this study was to apply different extraction techniques in separating sorghum leaf sheath polysaccharides,purify the polysaccharides by ion-exchange and gel-filtration chromatography,characterize the polysaccharide and establish its bioactive potentials.Ionic liquid（IL）,1-Alkyl-3-Methyliminidazolium Chloride（[C_nmim]Cl）,is one of the frontier extraction systems in the separation/purification of biomolecules.In this thesis,the[C₈mim]Cl/K₂CO₃ ionic liquid aqueous system showed the highest%extraction efficiency（%EE）in the range of 84-89%in the extraction of polysaccharides such as dextran,starch and cellulose.Dual frequency ultrasound-assisted extraction（DF-UAE）of crude polysaccharide(SL-C₈₀P)and[C₈mim]Cl/K₂CO₃ partially purified polysaccharide(SL-D₁₀P)of sorghum leaf sheath were achieved using response surface methodology（RSM）.The experimental yields for crude and partially purified polysaccharide,14.10%and 97.17%respectively,were found to be very close to the predicted,14.88%and 97.40%respectively.The extraction efficiency of Ionic liquid can be improved in the presence of ethanol.Among the IL’s,[C₄mim]Cl,[C₆mim]Cl,[C₈mim]Cl,[C₁₀mim]Cl,and[C₁₂mim]Cl,[C₈mim]Cl best formed immiscibility with K₂CO₃ in the presence of ethanol,displayed remarkably high%EE with a range of 84-95%and had lowest cellulose partition coefficient（0.21）.It was therefore selected as the working ternary system.Subsequently,response surface methodology was employed to simultaneously extract and separate partially purified polysaccharides from Sorghum bicolor L.leaf sheath using synergized ethanol adjunct-treated ionic liquid aqueous two phase system,[C₈mim]Cl/K₂CO₃,and dual frequency ultrasound.Under ultrasound conditions of 35 ^oC,20&60 KHz,25 min,an experimental yield of 16%w/w polysaccharides was achieved.Again,crude sorghum leaf sheath polysaccharide（39.99%wet matter（wm））wasobtained by subcritical ethanol-water（40%v/v）extraction（180 ^oC,40 min）.The subcritical extraction solution was transformed into an ionic liquid aqueous two-phase extraction system（[C₈mim]Cl/K₂CO₃）,and subsequently coupled with ultrasound extraction to obtain partially purified polysaccharides（PPP）.PPP yields of 20.89%,27.38%,and 36.49%（wm）were obtained using 60 k Hz,20/60 k Hz,and 20/40/60 k Hz ultrasound frequencies respectively.This part of the study determined the physicochemical characteristics and bioactive potentials of ultrasound-extracted purified polysaccharides（DFUP-1）from deep-freeze pretreated sorghum leaf sheath.Deep-freeze pretreatment induced breaks in the cell walls and resulted in a higher crude polysaccharide extraction yield of 14.35%compared to a yield of 9.23%without deep-freeze pretreatment.The crude extract of DFUP-1 was purified using DEAE-52 cellulose ion exchange and Sephadex G-100 gel-filtration chromatography.Ganoderma spp and Trametes versicolor bioactive metabolic products may be substrate dependent.This study tested the hypothesis that the polysaccharide products of Ganoderma-metabolized and Trametes versicolor-metabolized sorghum leaf sheath polysaccharides substrates would have enhanced bioactivity.Pretreated sorghum leaf sheath polysaccharide substrate was myceliated with either G.resinaceum（Gr）or G.enigmaticum（Ge）,or Trametes versicolor（Tv）for 15-20 days,dried and pulverized.The polysaccharide products for Ge-metabolized,Gr-metabolized and Tv-metabolized substrates,Ge SP,Gr SP,and Tv SP respectively,were extracted by ultrasound.The polysaccharide extracts were further purified using DEAE-52 cellulose ion-exchange and Sephadex G-100 gel filtration chromatography.Subsequently,three purified polysaccharide fractions of Ge SP（Ge SP-1,Ge SP-2 and Ge SP-3）,one fraction of Gr SP（Gr SP-1）and Tv SP（Tv SP-1）were further analyzed.The polysaccharide extract displayed interesting characteristics,antioxidant,cytotoxic and anti-inflammatory potential,and may have nutraceutical and pharmaceutical applications in industry.